The Hybridoma, NVS3 (Navy Vivax Sporozite 3) is deposited in the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, USA, by a deposit received Nov. 30, 1990, under the terms and conditions of the Budapest treaty for a period of thirty (30) years. The ATCC designation number is HB 10615, Under the terms of the deposit access to the culture will be available during pendency of the patent application to one determined by the Commissioner of Patents and Trademarks to those found to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122, and all restrictions on the availability to the public of the culture will be irrevocably removed upon grant of the Patent.
1. Field of the Invention
This invention relates to a passive protective agent against P. vivax. More particularly this invention relates to an antibody that, when a concentration of the antibody is injected intravenously, protects a subject to the limits of that concentration of antibody from developing malaria when the subject is subsequently challenged with live, infectious P. vivax sporozoites.
2. Description of the Prior Art
There have been major efforts toward development of malaria vaccines undertaken during the past 20 years. Although a commercially viable vaccine has not been achieved to the time this application is filed, there have been successes in providing vaccine protection. The continued vast investment in vaccine research by both governments world wide and industry shows an expectation of achieving a commercially viable vaccine. A commercially viable vaccine is one that provides protection with minimum side effects, is capable of being produced in quantity, and is stable in storage for a reasonable time under reasonable conditions. These conditions and requirements are well known in the medical and pharmaceutical arts. Even the near misses of total successes (e.g. successes with only a small population) are useful in understanding the mechanisms of malaria and further defining the parameters that will lead to a commercially successful vaccine or treatment. The current status of malaria vaccine development has been summarized in a recent Institution of Medicine Report1. The introduction to the section on vaccines is included verbatim to provide part of the background for this application.
Prospects for a Vaccine
Vaccination is an exceptionally attractive strategy for preventing and controlling malaria. Clinical and experimental data support the feasibility of developing effective malaria vaccines. For example, experimental vaccination with irradiated sporozoites can protect humans against malaria, suggesting that immunization with appropriate sporozoite and liver-stage antigens can prevent infection in individuals bitten by malaria-infected mosquitoes. In addition, repeated natural infections with the malaria parasite induce immune responses that can prevent disease and death in infected individuals, and the administration of serum antibodies obtained from repeatedly infected adults can control malaria infections in children who have not yet acquired protective immunity. These data suggest that immunization with appropriate blood-stage antigens can drastically reduce the consequences of malaria infection. Finally, experimental evidence shows that immunization with sexual-stage antigens can generate an immune response that prevents parasite development in the vector or, offering a strategy for interrupting malaria transmission.
Prospects for the development of malaria vaccines are enhanced by the availability of suitable methods for evaluating candidate antigens. These include protocols that allow humans volunteers to be safely infected with malaria, and the identification of many areas in the world where more than 75 percent of individuals can be expected to become infected with malaria during a three-month period. In contrast to vaccine for disease of low incidence, for which tens of thousands of immunized and non-immunized controls must be studied over several years, malaria vaccines could be evaluated in selected areas in fewer than 200 volunteers in less than a year.
Developments in molecular and cellular biology, peptide chemistry, and immunology provide the technological base for engineering subunit vaccines composed of different parts of the malaria parasite, an approach that was not possible 10 years ago. During the past 5 years, more than 15 experimental malaria vaccines have undergone preliminary testing in human volunteers. Although none of these vaccines has proven suitable for clinical implementation, progress has been made in defining the parameters of a successful vaccine and the stage has been set for further advancement.
Despite the inherent attractiveness and promise of this approach, there remain a number of obstacles to vaccine development. With the exception of the erthrocytic (blood) stages of P. falciparum, human malaria parasites cannot be readily cultured in vitro, limiting the ability of researchers to study other stages of this parasite and all stages of the other three human malaria parasite species.
In vitro assays, potentially useful for screening candidate vaccines for effectiveness, do not consistently predict the level of protective immunity seen in vivo. The only laboratory animals that can be infected with human malaria parasites are certain species of nonhuman primates, which are not naturally susceptible to these organisms. This makes it difficult to compare the results of many studies done in animals with what happens in human malaria infection.
The promises of modern vaccinology, while potentially revolutionary, have so far proved elusive. Few commercially available vaccines have been produced by this technology, for both scientific and economics reasons. Scientists have not yet been able to assemble defined synthetic peptides and recombinant proteins and combine them with new adjuvants and delivery systems into a practical human malaria vaccine. However, as discussed above and in the remainder of this chapter, there are good reasons to believe that this approach will ultimately succeed.
Approaches to Vaccine Development
The complex life cycle of the malaria parasite provides a number of potential targets for vaccination. Under investigation are vaccines that would be effective against the extracellular sporozoite, during the short period it spends in the bloodstream; the exoerythrocytic (or liver-stage) parasite, during the roughly seven days it develops within liver cells; the extracellular merozoite, released from liver cells or infected erthrocytes and free in the circulation prior to invading other erthrocytes; the asexual parasite that develops within red blood cells; exogenous parasite material released from infected erthrocytes; and the sexual-stage parasite, which occurs both inside erythrocytes and in mosquitoes. The optimal vaccine would include antigens from the sporozoite, asexual, and sexual stages of the parasite, thus providing multiple levels of control, but vaccines effective against individual stages could also prove highly useful. In addition, a vaccine against the Anopheles mosquito itself, which reduced the insect""s life span and prevented complete development of the parasite, could be valuable.
Regardless of the stage of parasite targeted for vaccine development, a similar strategy is envisioned. Based on knowledge of the mechanisms of protected immunity, specific parasite antigens (immunogens) are identified that induce a protective immune response, and synthetic or recombinant vaccines that accurately mimic the structure of that antigen are prepared.
In the subunit approach to vaccine development, this is done by combining the immunogen with carrier proteins, adjuvants, and live vectors or other delivery systems. This approach is being pursued throughout the world in laboratories studying infectious diseases. Clinical utility has yet to be demonstrated for the majority of these efforts, and barriers to obtaining satisfactory immunization by the subunit approach remain. Nevertheless, research on malaria subunit vaccines will continue to be at the cutting edge of this innovative and important approach to vaccine development.
It is clear from this description that major advances have been made, and many parasite proteins that could be targets of vaccine development have been identified. What has been lacking is an effective, economically feasible method for inducing protective immune responses against these already identified proteins. Perhaps the most striking example has been in the field of pre-erythrocytic stage malaria vaccine development in which there is already an effective vaccine for humans, the irradiated sporozoite vaccine, but the vaccine is totally impractical for widespread human use because of production and administration problems.
The Irradiated Sporozoite Model
In the 1940s, Mulligen and colleagues2 demonstrated that immunization of chickens with radiation attenuated Plasmodium gallinaceum sporozoite induced protective immunity. In the late 1960s, Nussenzweig and collegues3 demonstrated that immunization of A/J mice with radiation attenuated P. berghei sporozoite protected mice against challenge with infected erythrocytes were not protected. In the early 1970s Clyde and colleagues4-6 and Rieckmann and colleagues7,8 demonstrated that immunization of humans by the bite of irradiated Anopheles species mosquitoes carrying P. falciparum and in one case P. vivax sporozoites in their salivary glands protected these volunteers against challenge with live sporozoites. Like the immunity in mice, this immunity was stage specific, and it was also species specific; immunization with P. falciparum did not protect against P. vivax . However, it was not strain specific; immunization with P. falciparum sporozoites from Burma protected against challenge with sporozoites from Malaya, Panama and the Philippines4, and immunization with sporozoites from Ethiopia protected against challenge with a strain from Vietnam8. These human studies have been repeated recently9,10 reconfirming that there already is an effective malaria vaccine, and demonstrating this protective immunity lasts for at least 9 months11. Unfortunately, sporozoites have to be delivered alive, and since mature, infective sporozoites-infected mosquitoes, the targets and mechanisms of this protective immune response had to be identified so as to construct a synthetic or recombinant vaccine.
Of the four human malarias, P. vivax and P. falciparum are the most common and cause the majority of the malaria-induced disease seen worldwide. Prevention of infection by these human parasites would alleviate a major health problem in the tropical and subtropical areas of the world. The most promising method for the control of malaria appears to be the development and use of vaccines. One approach to malaria vaccine development involves the use of the circumsporozoite (CS) protein as a vaccine antigen. This protein covers the surface of the sporozoite. The sporozoite is the life stage of the parasite which is transmitted to humans by feeding female Anopheline mosquitoes. Evidence from both mouse and human malarias indicates that antibodies to the CS protein can provide protection in vivo against infection by sporozoites (Charoenvit et al., Infect. Immunity 55:604, 1987; Charoenvit et al., J. Immunol., 146, pp. 1020-1025, (1991). Khusmith et al., Science, 252, pp. 715-718, (1991).
In 1985, McCutchan and colleagues sequenced the gene for the CS protein in P. vivax and determined the amino acid sequence derived from that gene, (McCutchan et al., Science 230:1381, 1985). The monoclonal antibody originally used by McCutchan and colleagues (McCutchan et al., Science 230:1381, 1985) to identify the protein and isolate the nucleotide sequence which later became the subject of the McCutchan/Wistar U.S. Pat. No. 4,693,994 was originally developed by Charoenvit and Beaudoin of the Infectious Diseases Department, Naval Medical Research Institute (NMRI). This monoclonal antibody is the monoclonal antibody of this invention. It has been named or designated MAB Navy Vivax Sporozoite 3 (NVS3). In 1987, McCutchan and Wistar, in U.S. Pat. No. 4,693,994, described a repeated nine amino acid sequence within the CS protein as an immunodominant synthetic peptide. The repeated sequence is Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala.
In the ""994 patent and in other publications, McCutchan/Wistar maintain that the nine amino acid sequence is capable of inducing antibodies protective against P. vivax malaria. Experimental evidence indicates that while the McCutchan/Wistar sequence stimulates the development of anti-CS antibody in humans, it has not been shown to induce protective antibodies. In an article published in Am. J. Trop. Med. Hyg. 40(5), p455-464 (1989), Collins et al. describes tests in which Saimiri monkeys (Saimiri sciureus boliviensis), which are susceptible to human vivax malaria, were immunized with two different preparations (VIVAX-1 and NS181V20). Both preparations contain the McCutchan/Wistar peptide (Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). When these monkeys were challenged with 104 P. vivax sporozoites, there was no significant protection.
Nussenzweig et al., in U.S. Pat. No. 4,826,957, describes an immunogenic recombinant yeast expression product which contains a long sequence incorporating a portion of the P. vivax circumsporozite. The Nussenzweig et al. sequence contains multiple repeats of the sequence Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala as part of a complex polypeptide. When used as a vaccine, this polypeptide causes the formation of antibodies, the antibodies are directed at Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala and did not provide significant protection against challenge with sporozoites.
In U.S. Pat. No. 4,957,869, Arnot et al. describes an immunogenic peptide corresponding to P. vivax CS protein consisting of at least two repeats of the amino acid sequence Asp-Arg-Ala-X-Gly-Gln-Pro-Ala-Gly. X is defined as selected from the group consisting of Asp and Ala. The prior art approachs the problem from the premise that a vaccine is needed to provide protection against malaria. There is also a need for a simple material to protect against P. vivax. 
Accordingly, an object of this invention is a monoclonal antibody which provides passive protection against P. vivax. 
Another object of the invention is a pharmaceutical preparation which provides passive protection against P. vivax. 
An additional object of this invention is a means of providing temporary or limited protection against P. vivax by binding a particular site on the CS protein of P. vivax and thereby preventing infection by sporozoites of that parasite.
A further object of this invention is an agent to produce and isolate a human protective antibody against P. vivax. 
Yet an additional object of this invention is a method of using the unique binding and protective nature of the mouse monclonal antibody as a special reagent for conversion into a human monoclonal antibody which retains the same binding specificity and can therefore be used in humans to induce temporary antibody-mediated passive immunity.
Other objects and advantages of this invention will become clear as the detailed description of the present invention is presented. These and additional objects of the invention are accomplished by a murine, IgG3 monoclonal antibody designated NVS3 produced by immunizing mice with irradiated P. vivax sporozoites and pharmaceutical preparations of NVS3 which neutralize infectious sporozoites of P. vivax.